Mature ribosomal RNAs in all organisms are derived from long precursor molecules by post-transcriptional processing. In higher eukaryotes transcription of the rRNA precursor is initiated about 4 Kb 5' to the 5' end of mature 18S RNA and terminates at or near the 3' end of mature 28S RNA. The resultant 45S molecule has the order 5' yields 3': External transcribed spacer (ETS), 18S, internal transcribed spacer (ITS1), 5.8S, ITS2, 28S. While general pathways of processing of 45S in vivo have been elucidated, virtually nothing is known with regard to either the intrinsic features of the precursor molecule or the activities responsible for rRNA processing. To begin an investigation of this process, the human rRNA transcription unit has been subcloned into bacterial plasmids (SP6) capable of serving as efficient templates for in vitro transcription. Using labelled transcripts as substrates for in vitro processing reactions, accurate cleavage at the ETS/18S boundary is observed. This cleavage takes place inthe absence of protein and thus can be considered autocatalytic. However, the rate of cleavage is enhanced at least 50 fold by the addition of Hela cell nucleolar extract. The goals of this investigation are: (1) To establish both the primary sequence and secondary structure features of the precursor which promote accurate cleavage and (2) To define and characterize components of the nucleolar extract which are responsible for kinetic enhancement which defines the 5' end of 18S RNA. Subsequently, similar methodologies will be used to define parameters governing processing at the 3' end of 18S RNA, the 5' and 3' ends of 5.8S RNA and the 5' end of 28S RNA.